The cognitive-discursive model of evidentiality (authorization)

The paper discusses some functional aspects of evidentiality in scientific discourse and dialect speech in Russian. The aim is to show how evidentiality, traditionally called authorization in Russian, can be studied from functional and cognitive-discursive points of view. The authors propose a methodology of revealing additive meanings of evidential (authorizational) structures based on interpretation of subjective components of speech. The interpretation is based on the authorization model proposed by the authors

The ribosome-recycling step: consensus or controversy?

Ribosome recycling, the last step in translation, is now accepted as an essential process for prokaryotes. In 2005, three laboratories showed that ribosome-recycling factor (RRF) and elongation factor G (EF-G) cause dissociation of ribosomes into subunits, solving the long-standing problem of how this essential step of translation occurs. However, there remains ongoing controversy regarding the other actions of RRF and EF-G during ribosome recycling. We propose that the available data are consistent with the notion that RRF and EF-G not only split ribosomes into subunits but also participate directly in the release of deacylated tRNA and mRNA for the next round of translation

Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP

Ribosomes must dissociate into subunits in order to begin protein biosynthesis. The enzymes that catalyze this fundamental process in eukaryotes remained unknown. Here, we demonstrate that eukaryotic translocase, eEF2, which catalyzes peptide elongation in the presence of GTP, dissociates yeast 80S ribosomes into subunits in the presence of ATP but not GTP or other nucleoside triphosphates. Dissociation was detected by light scattering or ultracentrifugation after the split subunits were stabilized. ATP was hydrolyzed during the eEF2-dependent dissociation, while a non-hydrolyzable analog of ATP was inactive in ribosome splitting by eEF2. GTP inhibited not only ATP hydrolysis but also dissociation. Sordarin, a fungal eEF2 inhibitor, averted the splitting but stimulated ATP hydrolysis. Another elongation inhibitor, cycloheximide, also prevented eEF2/ATP-dependent splitting, while the inhibitory effect of fusidic acid on the splitting was nominal. Upon dissociation of the 80S ribosome, eEF2 was found on the subunits. We propose that the dissociation activity of eEF2/ATP plays a role in mobilizing 80S ribosomes for protein synthesis during the shift up of physiological conditions

The cognitive-discursive model of evidentiality (authorization)

The paper discusses some functional aspects of evidentiality in scientific discourse and dialect speech in Russian. The aim is to show how evidentiality, traditionally called authorization in Russian, can be studied from functional and cognitive-discursive points of view. The authors propose a methodology of revealing additive meanings of evidential (authorizational) structures based on interpretation of subjective components of speech. The interpretation is based on the authorization model proposed by the authors

New Structural Insights into Translational Miscoding

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Structural insights into the translational infidelity mechanism

International audienceThe decoding of mRNA on the ribosome is the least accurate process during genetic information transfer. Here we propose a unified decoding mechanism based on 11 high-resolution X-ray structures of the 70S ribosome that explains the occurrence of missense errors during translation. We determined ribosome structures in rare states where incorrect tRNAs were incorporated into the peptidyl-tRNA-binding site. These structures show that in the codon-anticodon duplex, a G·U mismatch adopts the Watson-Crick geometry, indicating a shift in the tautomeric equilibrium or ionization of the nucleobase. Additional structures with mismatches in the 70S decoding centre show that the binding of any tRNA induces identical rearrangements in the centre, which favours either isosteric or close to the Watson-Crick geometry codon-anticodon pairs. Overall, the results suggest that a mismatch escapes discrimination by preserving the shape of a Watson-Crick pair and indicate that geometric selection via tautomerism or ionization dominates the translational infidelity mechanism

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

International audiencePosttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA(Lys)(UUU) with hypermodified 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm(5)s(2)U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism

Accuracy mechanism of eukaryotic ribosome translocation

International audienceAbstract Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy 1 . Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon–anticodon interactions involving diphthamide 1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy 2 . The model demonstrates how the decoding centre releases a codon–anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a ‘pawl’ defining the directionality of translocation 3 . This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation